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The Effect Of Adding Chitosan Nanoparticles

To Casein Phosphopeptide Amorphous

Calcium Phosphate (Cpp-Acp) In Tooth

Remineralization: A Sem Study

FitriYunita Batubara*, Dennis*, Trimurni Abidin*, Harry Agusnar**

* Department of Conservative Dentistry, Faculty of Dentistry, University of Sumatera Utara Jl. Alumni No. 2 Kampus

USU, Medan. ** Department of Chemical Science Faculty of Mathematic and Natural Science, University of

Sumatera Utara Jl. Bioteknologi No. 1 Kampus USU, Medan.

Address for correspondence: Dr. FitriYunita Batubara, Department of Conservative Dentistry, Faculty of Dentistry, University of

Sumatera Utara Jl. Alumni No. 2 Kampus USU, Medan..

Abstract : With the advent of new materials, caries treatment is now carried out incontemporaryapproach. Based on the principle that caries can be remineralized, non-invasive intervention has been launched on caries lesion that has not formed into cavities, using therapeutic agents to heal the lesion by replacing the lost mineral in tooth structure. This study was aimed to compare the effect of CPP-ACP alone and the combination of CPP-ACP and chitosan nanoparticles in remineralizing tooth enamel by observing the morphological changes in enamel surface with Scanning Electron Microscope (SEM). Twenty four enamel samples from extracted molars were assigned into four groups. The first group was incubated in artificial saliva, the second was immersed in demineralizing solution, the third was immersed in demineralizing solution and applied with CPP-ACP gel, while the fourth group was immersed in demineralizing solution and applied with the combination of CPP-ACP gel and chitosan nanoparticles. The application of CPP-ACP and combination of CPP-ACP and chitosan nanoparticles were performed for 7 days. SEM was utilized to observe the morphological changes on enamel surface. The combination of CPP-ACP and chitosan nanoparticles resulted in littlemorphological enamel changes as compared to CPP-ACP group, and so it was concluded that both have the same ability to increase tooth enamel remineralization. Keywords: CPP-ACP, nanoparticle chitosan, enamel remineralization.

INTRODUCTION

The treatment of caries nowadays is done using

contemporary approach. The non-invasive

intervention for caries lesion that has not turned into

cavities is carried out using therapeutic agents to

heal the lesionby replacing the minerals lost due to

demineralization process. One of the methods to

reduce demineralization and increase enamel

remineralization is fluoride application, but

excessive doses can cause fluorosis during ages

when tooth development is occurring and in large

enough quantities can even be toxic.Taken together

the safety issues and dosage limitations are

considered an important limitation of fluoride

therapies thereby opening the window for the

development of new anti-cariogenic products with

fewer possible side-effects or that are potentially

more effective than fluoride.

It is now known that the penetration of calcium and

phosphate ions is essential in repairing deeper

damage in tooth structure. The newest technology in

ORIGINAL RESEARCH

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remineralization process is developed based

onphosphopeptides from casein protein of milk.1

Casein phosphopeptide (CPP) is comprised of

multiphosphoserylclusters with the ability to

stabilize calcium phosphate by forming colloidal

casein-phosphopeptide amorphous-calcium-

phosphate nanocomplexes(ACP). Through multiple

phosphoserylclusters, CPP binds to ACP into a

metastable solution which prevents the destruction

of calcium and phosphate ions.1

CPP-ACP also acts as a bioavailable reservoir of

calcium and phosphate ions, helping to maintain a

state of supersaturation of these ions, thereby

enhancing remineralization.1

The use of natural products is gaining popularityin

dentistry. Chitosan is one of the biomaterials which

recently being further developed due to its medical

benefits and safe-to-human use. Chitosan has special

features such as good biocompatibility, it is

biodegradable, non toxic, and does not cause

immunologic reactionor cancer. With these features,

chitosan and its modification with other agents can

be applied clinically as a biomaterial.2

The present study aimed to compare the effect of

CPP-ACP and the combinations of CPP-ACP and

chitosan nanoparticles in remineralizing tooth

enamel using Scanning Electron Microscope (SEM)

to observe the morphological changes in enamel

surface.

MATERIAL AND METHODS

Chitosan gel was created by dissolving 1gr chitosan

from the shell of blangkas (Tachypleusgigas) in 50

ml weak acid (acetic acid 1%), and then stirred in jar

test at 200 rpm for ±30 minutes until the gel was

formed. Whilst stirred, 20 drops of tripolyphosphate

solution was added into the gelto achieve smooth

appearance. The gel continued to be stirred in jar test

for another 30 minutes. The gel was subsequently

placed into ultrasonic bath to break the chitosan

particles into nanoparticles sizing 180 nm. This

residue ofnano chitosan was then added to CPP-

ACP (GC-Tooth Mousse) in 1:1 measurement.

In this study six molar teeth were used. The

rootswere removed using disc burr with water flow.

Each tooth was sectioned into four pieces in

mesiodistal and buccolingual/buccopalatal direction,

resulting in 24 samples which then were randomly

assigned into four groups. Buccal/lingual side was

mounted onparalon pipe of 1 cm in diameter with

acrylic resin, exposing an open area of 2x2 mm.

Samples were numbered 1 to 24 and randomly

assigned to four groups; six samples for each group.

The first group was only incubated in artificial saliva

at 37°C. The second group was immersed in

demineralizing solution for 4 days and then

incubated in artificial saliva at 37°C. The third group

was immersed in demineralizing solution for 4 days,

applied with CPP-ACP gel once daily for 5 minutes,

and then incubated in artificial saliva at 37°C. The

last group was immersed in demineralizing solution

for 4 days and applied with the combination gel of

CPP-ACP and chitosan nanoparticles for 5 minutes

once a day, and then incubated in artificial saliva at

37°C. The whole procedure took seven days. The

SEM observation of samples was done on the eight

day.

RESULTS

Scanning Electron Microscope (SEM) was utilized

to observethe morphological features on enamel

surface of every treatment groups. The enamel under

observation in this study was a clinically and

macroscopically intact surface which did not

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undergo polishing. The results were depicted in the

following pictures: (A) morphological appearance of

enamel surface which was only incubated in

artificial saliva, (B) morphological appearance of

enamel surface which underwent demineralization,

(C) morphological appearance of enamel surface

which underwent demineralization and application

of CPP-ACP gel, and (D) morphological appearance

of enamel surface which underwent

demineralization and application of CPP-ACP-

chitosan gel.

Fig.1: The morphological appearance of enamel surface under

SEM. a) The enamel was only incubated in artificial saliva; b)

The enamel was immersed in demineralizing solution; c) The

enamel was applied with CPP-ACP gel; d) The enamel was

applied with the combination gel of CPP-ACP and chitosan

(magnification 1000X).

DISCUSSION

The samples in the present study were crown of

molars extracted from patients aged 18-35 years, in

good and intact condition clinically and

macroscopically. Each crown was divided into four

parts, mesiobuccal, mesiolingual, distobuccal and

distolingual, and then assigned to group I, II, III and

IV, respectively. All mesiobuccal part would be in

group I, mesiolingual in group II, distobuccal in

group III, and distolingual in group IV. This was

done based on the author’s assumption that enamels

parts from the same teeth would inhibit the same

appearancebefore treatment.

The enamel analyzed in this study was

macroscopically intact enamel which did not

undergo polishing. Qualitative analysis was done

using SEM to observe morphological appearance of

each group.

In the first group which was incubated in artificial

saliva (Figure (a)), it was found that the enamel

surface wasnot entirely smooth and enamel tip was

observed as a normal variety of enamel.

Figure (b) illustrated the appearance of enamel

which was immersed in demineralizing solution.

Porosity and wavy surface were noted, indicating

demineralization process that had taken place.

Figure (c)showed enamel surface which underwent

demineralization and application of CPP-ACP gel.

Less porosity and smoother surface was seen

compared to enamel in the second group (Figure

(b)). Several studies have reported that CPP-ACP

possess the ability to penetrate further into enamel to

replace calcium and phosphate ions lost to

demineralization. The deeper penetration resulted in

more optimum ion replacement, hence maximizing

enamel structure repair.3-5

Figure (d) displayed enamel surface applied with the

combination of CPP-ACP and chitosan. The enamel

surface became much smoother compared to enamel

applied with CPP-ACP only (Figure C). It was

assumed to be related with the ability of chitosan to

prevent the loss of calcium and phosphor during

enamel demineralization process.6

The nano-sized chitosan particles increase the

surface area up to hundred times compared to micro-

a b

d c

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sized particles, therefore increasing the efficacy of

chitosan in binding with other chemicalclusters.7

The result of the study showed that CPP-ACP and

the combination of CPP-ACP and chitosan have the

same ability to increase tooth enamel

remineralization.

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Remineralization of enamel subsurface lesion in situ

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6. Sugita P, Wukirsari T, Sjahriza A, Wahyono

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How to cite this article:

Batubara FY, Dennis, Abidin T, Agusnar

H. The Effect Of Adding Chitosan

Nanoparticles To Casein Phosphopeptide

Amorphous Calcium Phosphate (Cpp-

Acp) In Tooth Remineralization: A Sem

Study. IJRD 2015;4(1):6-9.